Histone H3 lysine 9 and H4 lysine 20 trimethylation and the expression of Suv4-20h2 and Suv-39h1 histone methyltransferases in hepatocarcinogenesis induced by methyl deficiency in rats.

The field of cancer epigenetics has received much attention in recent years. However, the relationship of cancer epigenetics with cancer etiology is not clear. Recent studies suggest the involvement of altered DNA methylation and histone modifications in the emergence of epigenetically reprogrammed cells with specific tumor-related phenotypes at premalignant stages of tumor development. In this study, we used a methyl-deficient model of rodent hepatocarcinogenesis to examine the roles of DNA, histone H3 lysine 9 and histone H4 lysine 20 methylation, and the level of the expression of Suv39h1 and Suv4-20h2 histone methyltransferases in the carcinogenic process. We demonstrated that the development of liver tumors was characterized by progressive demethylation of DNA repeats, decrease in histone H4 lysine 20 trimethylation, and a gradual decrease in the expression of Suv4-20h2 histone methyltransferase. A prominent increase in the trimethylation of histone H3 lysine 9 and in the expression of Suv39h1 histone methyltransferase was observed in preneoplastic nodules and liver tumors indicating the promotional role of these epigenetic alterations at later stages of carcinogenesis. The appearance of tumor-specific epigenetic alterations (demethylation of repetitive elements, loss of histone H4 lysine 20 trimethylation, altered expression of Suv4-20h2 and Suv39h1 histone methyltransferases) at preneoplastic stages of hepatocarcinogenesis provides experimental support for the epigenetic hypothesis of tumorigenesis that considers stress-induced epigenetic reprogramming of the cell as an important prerequisite to succeeding mutations.

Irreversible global DNA hypomethylation as a key step in hepatocarcinogenesis induced by dietary methyl deficiency.

Dietary methyl group deprivation is now well recognized as a model of hepatocarcinogenesis in rodents. In the present study, we examined the effects of feeding a methyl-deficient diet followed by a methyl-adequate diet on the extent of methylation of liver DNA and on the formation and evolution of altered hepatic foci. Male F344 rats were fed a methyl-deficient diet for 9, 18, 24, and 36 weeks, followed by re-feeding a methyl-adequate diet for a total of 54 weeks. Similar to previous findings, the methyl-deficient diet resulted in decreased levels of S-adenosylmethionine (SAM), SAM/SAH ratios, and global DNA hypomethylation. Feeding the methyl-adequate diet restored the liver SAM levels and SAM/SAH ratios to control levels in all experimental groups. In contrast, re-feeding the complete diet restored DNA methylation to normal level only in the group that had been fed the methyl-deficient diet for 9 weeks; in animals exposed to methyl deprivation longer, the methyl-adequate diet failed to reverse the hypomethylation of DNA. Liver tissue of rats exposed to methyl deficiency for 9, 18, 24, or 36 weeks was characterized by the persistent presence of placental isoform of glutathione-S-transferase (GSTpi)-positive lesions despite re-feeding the methyl-adequate diet. The persistence of altered hepatic foci in liver after withdrawal of methyl-deficient diet serves as an indication of the carcinogenic potential of a methyl-deficient diet. Substitution of the methyl-deficient diet with complete diet failed to prevent the expansion of initiated foci and restore DNA methylation in animals exposed to deficiency for 18, 24, or 36 weeks. The association between DNA hypomethylation and expansion of foci suggests that stable DNA hypomethylation is a promoting factor for clonal expansion of initiated cells. These results provide an experimental evidence and a mechanistic basis by which epigenetic alterations may contribute to the initiation and promotion steps of carcinogenesis.

Saratin (an inhibitor of platelet-collagen interaction) decreases platelet aggregation and homocysteine-mediated postcarotid endarterectomy intimal hyperplasia in a dose-dependent manner.

BACKGROUND: This study investigated Saratin's (Merck KGaA, Darmstadt, Germany) prevention of platelet adhesion and intimal hyperplasia at different doses and in the hyperhomocystinemia rat carotid endarterectomy (CEA) model. METHODS: Rats were divided into two groups: (1) platelet adhesion or (2) luminal stenosis because of intimal hyperplasia. At CEA, rats received 0, 0.5, 5.0, 10.0, or 20.0 microg Saratin on the artery. Post-CEA platelet aggregation was evaluated by standard error of the mean. Intimal hyperplasia group received either (1) control or (2) 4.5 g/kg DL-homocystine diets for two weeks followed by CEA and treated with diluent or 5.0 microg Saratin. Endpoints included platelet adhesion, intimal hyperplasia, plasma homocysteine (HCys), and its metabolic enzymes cystathionine beta-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR). RESULTS: Platelet adhesion: post-CEA, platelet adhesion was reduced by 63%, 67%, and 67% in Saratin doses > or =5.0 microg. Intimal hyperplasia: 5.0 microg Saratin in the HCys group decreased intimal hyperplasia by 45% compared with the non-Saratin-treated HCys group. Plasma HCys levels were not altered with Saratin treatment in the HCys groups nor were CBS or MTHFR. CONCLUSIONS: Saratin significantly inhibited platelet adhesion at > or =5.0 microg, and Saratin at 5.0 microg attenuated luminal stenosis in a hyperhomocysteinemic rat CEA model.

Plasma homocysteine measurements after carotid artery manipulation and clamping in a rat CEA model.

OBJECTIVE: The effect of a rat carotid endarterectomy (CEA) on homocysteine and the metabolic enzymes methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS) was studied. METHODS: Rats were placed into 7 groups: (1) no anesthesia (NA), (2) anesthesia only (AO), (3) skin opened and closed (O/C), (4) skin opened with exposure of the carotid artery and closed (O/E/C), (5) carotid isolated and clamped (CO), (6) open CEA, and (7) open femoral endarterectomy (FEA). End points included homocysteine, hepatic MTHFR, and CBS activity. RESULTS: Homocysteine in the NA, AO, O/C, O/E/C, and FEA were low and not different. CEA produced a 6-fold increase in homocysteine when compared with non-CEA groups. Specifically, CEA produced an increase in homocysteine versus the AO group at 2 weeks (11.3 +/- 0.7 vs 2.1 +/- 0.9 micromol/L;P < .001), 4 weeks (8.9 +/- 0.7 vs 3.5 +/- 0.9 micromol/L; P = .004) and 6 weeks (7.7 +/- 0.9 vs 3.1 +/- 1.5 micromol/L; P = .03). The CO group had increased homocysteine versus the O/C, O/E/C, and FEA, but was lower than the CEA group. CEA produced an increase in MTHFR and CBS versus the AO group. CONCLUSIONS: CEA resulted in elevated levels of homocysteine; however, when broken down into its component parts, no elevation was observed except for a small increase with the CO procedure. Manipulation of the femoral artery did not raise homocysteine levels. The increase in homocysteine is possibly due to the combination of vessel wall damage and changes in cerebral blood flow dynamics.

Cigarette smoke increases intimal hyperplasia and homocysteine in a rat carotid endarterectomy.

BACKGROUND: Homocysteine and smoking are independent risks for CVD; however their importance in post-CEA intimal hyperplasia is unclear. We performed a CEA in rats exposed to cigarette smoke with the hypothesis that smoking would increase intimal hyperplasia that may be associated with an elevated serum homocysteine. Folic acid (FA) and the homocysteine metabolic enzymes MTHFR and CBS were used to test for the significance of homocysteine elevation. MATERIALS AND METHODS: Rats underwent an open CEA. N = 13 rats received smoke exposure 2 weeks prior, and 2 weeks post-CEA and N = 12 received no smoke. Each group was divided into either control or an FA-added diet resulting in four groups. Rats were sacrificed at 2 weeks post-CEA; liver, urine, blood, and carotid arteries samples were obtained. RESULTS: Smoked rats had increased urinary peak and trough cotinine levels versus non-smoke rats, which decreased with FA. Smoke exposure increased intimal hyperplasia versus non-smoke controls by nearly 120% (57.8 +/- 6.2 versus 26.8 +/- 5.4% luminal stenosis, P = 0.005). Smoke-exposed rats had an increased serum homocysteine versus non-smoke controls (8.3 +/- 0.8 versus 5.7 +/- 0.8 microm, P = 0.014). Smoked rats given FA had decreased serum homocysteine compared to the smoke group. Along with reductions in homocysteine, FA eliminated the increase in intimal hyperplasia seen with smoke exposure (33.5 +/- 6.1 versus 57.8 +/- 6.2% luminal stenosis, P = 0.03). CBS activity decreased in smoked rats by nearly 20% versus non-smoke rats. FA supplementation in smoked rats both (1) increased CBS activity and (2) decreased MTHFR compared to control non-smoke-exposure levels. CONCLUSION: Smoking increases plasma homocysteine and post-CEA intimal hyperplasia. This suggests homocysteine has an etiological role in the intimal hyperplasia increase observed with smoking, since both were negated with FA.

Intimal hyperplasia following carotid endarterectomy in an insulin-resistant rat model.

Hyperhomocysteinemia, a known risk factor for cardiovascular disease, results in an elevation of intimal hyperplasia (IH) following a carotid endarterectomy (CEA) in a rat model. An exaggerated IH response following CEA has been observed in rats with dietary induced hyperhomocysteinemia. Type 2 diabetics often present with hyperhomocysteinemia and are at higher risk for developing vascular blockage following surgical procedures. To determine if insulin resistance increases IH risks following endarterectomy, the 3 goals of this study were: (1) to establish plasma homocysteine concentrations in dietary induced insulin-resistant rats and their controls, (2) to investigate whether a positive correlation of IH and plasma homocysteine response occurs following CEA in the insulin-resistant rat model, and (3) if so, to attempt to decrease IH by supplementation with folic acid, a known enzymatic cofactor in the homocysteine metabolic pathway. To achieve these aims, male rats (275 to 300 g) were fed 1 of 4 diets for a 4-month period: (1) high-fat sucrose (HFS), (2) low-fat complex carbohydrate (LFCC), (3) HFS + 25 mg/kg folic acid (HFS+F), or (4) LFCC + 25 mg/kg folic acid (LFCC+F). At the end of the 4-month period the rats underwent an open (non-balloon) unilateral CEA. Two weeks post-endarterectomy, blood, liver and carotid tissue were removed to measure plasma insulin, folic acid, and homocysteine, 2 key enzymes of homocysteine metabolism-methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS)-and percent lumenal stenosis (IH%). Computer-assisted morphometric analysis was used to measure the percentage of IH in the carotid artery. Plasma homocysteine was significantly higher in the HFS group when compared with the LFCC group (11.3+/-1.3 micromol/L v 7.4+/-0.6 mircomol/L, P=.008) as was post-endarterectomy IH producing lumenal stenosis (30.7%+/-4.2% v 14.0%+/-4.3%, P=.008). Plasma insulin in the HFS group was higher than the LFCC (control) group and was significant (36.3+/-3.0 microU/mL v 21.1+/-0.8 microU/mL, P=.0004). Folic acid supplementation in the HFS group resulted in reductions of plasma homocysteine (HFS v HFS+F, 11.3+/-1.3 micromol/L v 7.95+/-1.0 micromol/L, P=.02) and post-endarterectomy IH (HFS v HFS+F, 30.7%+/-4.2 % v 10.4%+/-1.6%, P=.0001). The control or LFCC group was not statistically different from the HFS+F group in homocysteine or IH. Folate supplementation did not decrease insulin concentrations in the HFS+F group compared to the LFCC group. We conclude that the HFS diet produced an insulin-resistant state with an elevated plasma homocysteine and an exaggerated IH response following carotid endarterectomy in this rat model. Dietary folate supplementation reduced plasma homocysteine concentrations in the HFS diet, which implicates hyperhomocysteinemia as an etiologic factor in the development of post-CEA IH in this insulin-resistant rat model.

S-adenosyl-L-methionine is able to reverse micronucleus formation induced by sodium arsenite and other cytoskeleton disrupting agents in cultured human cells.

Deficiencies of folic acid and methionine, two of the major components of the methyl metabolism, correlate with an increment of chromosome breaks and micronuclei. It has been proposed that these effects may arise from a decrease of S-adenosyl-L-methionine (SAM), the universal methyl donor. Some xenobiotics, such as arsenic, originate a reduction of SAM levels, and this is believed to alter some methylation processes (e.g. DNA methylation). The aim of the present work was to analyze the effects of exogenous SAM on the micronucleus (MN) frequency induced by sodium arsenite in human lymphocytes treated in vitro and to investigate whether these effects are related to DNA methylation. Results showed a reduction in the MN frequency in cultures treated with sodium arsenite and SAM compared to those treated with arsenite alone. To understand the mechanism by which SAM reduced the number of micronucleated cells, its effect on MN induced by other xenobiotics was also analyzed. Results showed that SAM did not have any effect on the increase in MN frequency caused by alkylating (mitomycin C or cisplatin) or demethylating agents (5-azacytidine, hydralazine, ethionine and procainamide), but it reduced the number of micronucleated cells in those treated with agents that inhibit microtubule polymerization (albendazole sulphoxide and colcemid). Since albendazole sulphoxide and colcemid inhibit microtubule polymerization, we decided to evaluate the effect of SAM on microtubule integrity. Data obtained from these evaluations showed that sodium arsenite, albendazole sulphoxide, and colcemid affect the integrity and organization of microtubules and that these effects are significantly reduced when cultures were treated at the same time with SAM. The data taken all together point out that the positive effects of SAM could be due to its ability to protect microtubules through an unknown mechanism.

The prospective role of abnormal methyl metabolism in cadmium toxicity.

Several lines of evidence point to the probable role of abnormal methylation processes in the toxicology of metals and other xenobiotics. The spectrum of toxic effects exhibited by such metals as Ni, As, and Cd, as well as by Zn deficiency, often resemble those seen in animals chronically fed methyl-deficient diets. These metal-associated pathologies include cancer, atherosclerosis, birth defects, neurological disturbances, and pancreatic lesions. In addition, each of the above agents has been shown to alter normal methyl group metabolism in vivo or in vitro. In the present studies, we compared the effects on the enzyme DNA methyltransferase (MTase) of two metal ions: the essential metal Zn and the carcinogen Cd. MTase extracts were obtained from the hepatic nuclei of rats fed a methyl-deficient diet (lacking choline and folate) for 7 and 24 weeks. Control animals were fed the same diet supplemented with each of these vitamins. Zn and Cd both inhibited MTase in the nuclear extracts from both the control and the methyl-deficient rats. The inhibitory activity of Cd was greater than that of Zn regardless of whether the nuclear extracts were from the control or the deficient animals. In addition, the kinetics of Cd inhibition of MTase activity were different in the nuclear extracts from the control and methyl-deficient rats. The results provide evidence that the carcinogenic effects of Cd may be mediated in part through abnormal DNA methylation.

The effects of diet, genetics and chemicals on toxicity and aberrant DNA methylation: an introduction.

In the early 1930s, the group of Banting and Best showed that the choline moiety of lecithin was responsible for the prevention of the fatty livers produced in pancreatectomized dogs treated with insulin. This was the first study linking abnormal methyl metabolism with disease. Since then, deficiencies of each of the four essential dietary sources of methyl groups (choline, methionine, vitamin B-12 and folic acid) have been associated with increased risk of a number of diseases. Choline-deficient diets were shown to enhance liver tumor formation in rats, and such diets frequently were found to lead to atherosclerosis. Although methionine deficiency per se was not extensively studied in vivo, its metabolic antagonist ethionine did cause liver cancer and pancreatic toxicity in rodents. Deficiencies of vitamin B-12 and of folic acid have long been shown to cause neurological disturbances and birth defects both in humans and in experimental animals. In 1969 inborn errors of metabolism leading to the accumulation of the demethylated metabolite of methionine, homocysteine, were proposed as contributing to the early onset of atherosclerosis. Before 1990, numerous studies described the abnormal methylation of DNA in tumors and transformed cells. Less frequently investigated, however, were the exogenous and endogenous agents leading to such abnormal methylation. These included genetic variants among rodent strains and the methyl-deficient diets that caused liver cancer. In addition, several chemicals, particularly carcinogens, were shown to alter DNA methylation. The possible links between chemically induced alterations in DNA methylation and development of other diseases were little explored. However, by 1990, a chain of causality had been established in experimental carcinogenesis linking dietary methyl deficiency with methyl insufficiency in vivo, as well as with the abnormal methylation of DNA and of specific genes. Also during this period, the diminished activity of the enzyme methylenetetrahydrofolate reductase (EC 1.5.1.20), which is responsible for the actual de novo synthesis of methyl groups, was shown to be associated with increased risk of developing atherosclerosis, neurological disorders and birth defects. The exponential rise in studies on methyl metabolism and DNA methylation since then enables us to examine here the extent to which the mechanisms by which abnormal methylation processes seem to exert their toxic effects in one disease may be applicable to other pathologies.

The effect of troglitazone on plasma homocysteine, hepatic and red blood cell S-adenosyl methionine, and S-adenosyl homocysteine and enzymes in homocysteine metabolism in Zucker rats.

We studied the effect of troglitazone on the plasma concentrations of homocysteine (tHcy), the erythrocyte and hepatic concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and the hepatic activities of cystathionine-beta-synthase (C beta S) and methylenetetrahydrofolate reductase (MTHFR) in lean and fatty Zucker rats (a model of insulin resistance). Four groups of female Zucker rats were studied. Troglitazone (200 mg/kg) was administered by gavage daily for 3 weeks to lean and fatty Zucker rats. The other 2 groups served as controls. The blood parameters were determined at days 0, 10, and 21. The hepatic SAM and SAH concentrations and MTHFR and C beta S were measured in the 3-week liver samples. Plasma homocysteine fell significantly in all troglitazone-treated animals from a mean +/- SD of 7.6 +/- 1.5 micromol/L to 4.5 +/- 1.1 micromol/L (P <.02) but not in control animals (5.7 +/-1.8 micromol/L to 5.9 +/- 1.8 micromol/L). The decreases induced by troglitazone in homocysteine were seen in both the lean and the fatty Zucker rats. This was accompanied by significant rises in the hepatic concentrations of SAH and SAM + SAH. In addition, a significant decline in the hepatic SAM/SAH ratio was observed. The mean +/- SD hepatic C beta S (expressed as nmol of cystathionine formed at 37 degrees C) in the troglitazone-treated rats was 1,226 +/- 47 nmol/h/mg protein, which was significantly higher than that in the control group (964 +/- 64 nmol/h/mg protein; P =.03). We conclude that troglitazone lowers plasma homocysteine in insulin-resistant animals. The homocysteine-lowering effects of troglitazone may be mediated in part by a shift in the concentrations of tHcy and its related metabolites from the blood to the liver as well as by an upregulation of hepatic C beta S activity. These data support the hypothesis that insulin may regulate homocysteine metabolism through regulation of hepatic C beta S activity, although activity of other hepatic enzymes not studied here may also contribute to these observations.

Methamphetamine Treatment Affects Blood and Liver S-Adenosylmethionine (SAM) in Mice: Correlation with Dopamine Depletion in the Striatuma.

Methamphetamine (METH) is a major drug of abuse which causes neurotoxicity by depleting dopamine, its metabolites, high-affinity dopamine uptake sites and tyrosine hydroxylase activity in the striatum. Dopamine depletion and reduced dopamine transit are associated with depression. S-Adenosylmethionine (SAM) is the chief methyl donor used in dopamine and other neurotransmitter metabolism in mammals. Low SAM is associated with depression and other psychological and neurological disorders in humans. SAM is used to treat depression and some other neurological and psychiatric disorders. The present study was designed to determine if single or multiple doses of METH induce alterations in blood or liver SAM in mice and if these correlate with dopamine levels in the striatum. Adult male C57 mice were injected intraperitoneally with either single (1 × 40 mg/kg) or multiple (4 × 10 mg/kg) doses of METH. Animals were sacrificed at various intervals. A single injection of METH resulted in slightly higher blood SAM levels at 4 hr. Multiple doses of METH resulted in decreased hepatic and blood SAM levels at 72 hr. Blood SAM returned to control levels by 1 wk. Published work shows that dopamine levels increase hours after a single injection of METH, whereas dopamine decreases days after multiple injections of METH. These present data clearly demonstrate that METH dosing leads to significant alterations in liver and blood SAM and that these changes in SAM levels correlate with changes in striatal dopamine levels.